Spore-forming microorganisms belonging to aerobic Bacillus species and obligate anaerobic Clostridium species, which are naturally occurring in agricultural raw materials harvested from the soil, such as vegetables and spices, form highly heat-resistant endospores, and thus serve as direct or indirect causes of deterioration of the quality of processed food products and threaten food hygiene.
The spore core is maintained in a dehydrated and dried state, and is surrounded by a peptidoglycan layer called the cortex. The cortex is encased in the coat layer composed largely of proteins. Due to the specialized structure and biochemical properties, spores are highly resistant to heat, chemical reagents such as antimicrobial substances or antibiotics, lysozymes, physical impacts, UV radiation, high pressure, high voltage pulse electric fields, and the like. Spores can remain dormant for long periods of time to survive in unfavorable growth environments. Thus, sterilization conditions for spores must be primarily considered to ensure the microbial safety of processed food products.
In addition, spore-forming microorganisms, which are problematic because they are naturally occurring in soil-derived food raw materials, such as vegetables and spices, are significant risk factors in the sterilization of retort products charged into various types of containers, such as cans, pouches and trays. That is, spore-forming microorganisms are secondary contaminants resulting from the generation of cold points, at which sufficient heat treatment of over Fo 4 is not achieved, and problems with packaging materials, such as pin holes, and thus are major sterilization targets.
It is therefore important to research and develop germination inhibition and sterilization methods of spore-forming microorganisms in order to ensure the hygiene and shelf life of processed food products.
Since pathogens not forming spores have weak resistance to heat and low resistance to chemical treatment, they are sufficiently inhibited in growth or killed by heat treatment at less than 100° C. or by treatment with commercialized antimicrobial agents, such as organic acids, alcohol and bacteriocine. In contrast, spores are not easily killed due to their structural, chemical and ecological properties. Thus, heat-resistant spores are typically killed using a retort sterilization method based on heating at a high temperature of 121° C. under a high pressure of 1-1.5 Kg/cm2 for a period ranging from a few minutes to several tens of minutes. However, such high temperature treatment significantly damages sensory qualities including taste, appearance and texture, and destroys a lot of nutrients, thereby impeding the development of high-quality processed food products. Another commercial sterilization method involves indirect sterilization, which is based on inducing germination of spores at a mild temperature of 65° C. for 5 to 6 hours to convert spores into vegetative cells and performing sterilization. This method has an advantage of allowing sterilization of heat-resistant spores at temperatures lower than 100° C. without quality reduction, but has some drawbacks including the fact that it is time-consuming when applied to industrial production and entails a high risk of microbial contamination in summer. Also, methods of inhibiting spore germination are used, which employ sodium lactate, lysolecithin, poly fatty acid ester, L-phenylalanine, essential oils, glycine, L-serine, and the like. However, since these methods using food additives have only inhibitory effects on spore germination and no spore-killing effect, potential risk factors remain.
In addition, studies have been performed for various antimicrobial compositions.
According to Alkhayat, Huhtanen, Ueda, et al., hot water and ethanol extracts of spices, such as clove, mace, white and black pepper, laurel, and nutmeg, have growth inhibitory effects on spores of Bacillus botulinus types A and B at a minimal inhibition concentration (MIC) of 125 μg/ml. According to Hara et al., tea extracts, such as tannine, polyphenol, theaflavin and catechin, are effective in inhibiting the germination of spores. Also, caffeic acid and protamine, which is a highly-basic protein (peptide) binding to DNA in the nuclei of the sperm of fish, are effective in killing spores and inhibiting spore germination. Protamine kills vegetative cells of Bacillus species by damaging the cell wall and the plasma membrane, and has growth inhibitory effects on spores of Bacillus species through the inhibition of DNA, RNA and protein synthesis and respiration inhibition in the ATP level. Also, the spore death may be greatly stimulated by protamine's cooperative action with heat. In addition, other natural antimicrobial materials, which are known to have an inhibitory effect on spore germination and a sterilization effect on spores, include the following: polylysine, which has a growth inhibitory effect by serving as a surfactant affecting the spore structure; peptides and proteins consisting of amino acids, such as bacteriocine, nisin and pediocines, which do not directly affect dormant spores, but affects the core of germinating spores by penetrating the thin membrane of spores at the early swelling stage of spore germination, thereby inhibiting propagation; and ethanol, which has a growth inhibitory effect during spore germination or sporulation. However, most antimicrobial agents against spores have a growth inhibitory action rather than a direct sterilization effect against dormant spores, or display only growth inhibitory action during spore germination or sporulation. Also, substances having direct sterilization effects display an unsatisfactory spore-killing effect of about 101.
A review of related domestic and foreign literature yields the following. Korean Pat. Application No. 1992-18019 discloses a composition having an inhibitory effect on spore germination, comprising an extract from a mixture of defatted starch of a plant species belonging to the genus Ricinius and roots of another plant species belonging to the genus Coptis, and states that berberine is a key substance in the inhibition of spore germination. Korean Pat. Application No. 1996-7000557 discloses a method and a composition for killing or inhibiting the growth of yeast or spore-forming microorganisms by contacting the microorganisms, in the presence of a peroxide and chloride or bromide, with a haloperoxidase and at least one antimicrobial activity enhancing agent. This patent describes that the antimicrobial activity enhancing agent includes certain alpha-amino acids, and are preferably compounds having a structure which contains hydrogen, an unsubstituted or hydroxyl- or amino-substituted, straight or branched chain alkyl group having from 1 to 6 carbon atoms, or an unsubstituted or hydroxyl- or amino-substituted arylalky group having from 7 to 12 carbon atoms. This patent also describes that the antimicrobial activity enhancing agents include alpha-amino acids selected from the group consisting of glycine and l- or d-enantiomers of alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine and tyrosine, and alkyl esters thereof. Japanese Pat. Application No. 1995-72164 discloses a diglycerine fatty acid monoester composition for inhibiting the germination and propagation of heat-resistant spores which cause some problems in beverage processing, comprising fatty acids, such as lauric acid, myristic acid and palmitic acid, and monoester. Japanese Pat. Application No. 1993-301163 discloses a method of inducing inhibition of spore germination using a composition comprising fatty acids including glycerine monostearic acid ester and isolecithin. The aforementioned inventions mainly aim to inhibit the growth of spores, and most antimicrobial materials described in these inventions cab be considered synthetic food additives rather than natural antimicrobial materials.
Based on this background, the intensive and through research into the development of natural antimicrobial agents having an effect of completely sterilizing spores with no side effects resulted in the finding that among one hundred edible plant materials including herbal spices, Chinese herbal medicines, tropical fruits and vegetables, an extract of Torilidis Fructus has a very strong sterilization effect on spores of Bacillus subtilis, thereby leading to the present invention.